The general objectives of this project are to develop analogues of acyl-CoA that are active-site-directed irreversible inhibitors and to use these analogues to probe structural and functional features of certain enzymes that accept acyl-CoA as a substrate. Both classical affinity labels and photoaffinity labels are under investigation. The general approach in the synthesis of classical affinity labels is to condense one end of a bifunctional cross-linking reagent (e.g. 1,3-dibromopropanone) with the thiol of CoA. Photoaffinity labels are made by incorporating an aryl azide in the structure of the acyl-CoA analogue. We intend to synthesize highly radioactive reagents by phosphorylating chemically synthesized 3'-dephospho analogues with (32P)-ATP using 3'-dephospho-CoA kinase. The photochemistry of the aryl azides used in the analogues will be characterized so as to provide rational guidelines in designing "optimal" reagents for photolabeling experiments. The enzymes of interest this project period are short chain acyl-CoA hydrolase from beef kidney and membrane-bound enzymes that utilize long-chain acyl-CoA. For the latter studies the analogue of long-chain acyl-CoA will be photoactivatable. The critical micelle concentration of this analogue will be measured by a novel technique, and its general properties with regard to interacting with phospholipids and proteins will be tested prior to exploring the photoaffinity labeling of membranes.